Methods and materials for microorganism capture

ABSTRACT

Material complexes that capture biologicals and methods of synthesizing and using such complexes composed of fluid-insoluble material and a receptor are provided herewith. The fluid-insoluble material has reactive functionality on its surface, including hydroxyl, amino, mercapto or epoxy functionality material. The material can be agarose, sand, textile, or any combination thereof. The receptor is selected from the group consisting of mono- and poly-saccharides, heparin, or any combination thereof. Also provided are methods whereby releasing the captured biologicals is controllable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application of PCTApplication No. PCT/US2014/026540, filed Mar. 13, 2014, which claimspriority to U.S. Provisional Application No. 61/784,432, filed Mar. 14,2013, each of which is incorporated herein by reference in its entirety.

FIELD

The present disclosure is directed to fluid-insoluble material complexesthat control the biological composition of fluids, and methods ofmanufacturing and using the same.

BACKGROUND

Affinity ligand-matrix conjugates for use as chromatography adsorbentsfor protein purification are well established. For example, the use ofphenyl-based ligand for protein purification, including purification ofthe in-demand monoclonal antibodies has been disclosed in the patentliterature. Other known affinity ligands used in chromatography media topurify influenza A are fusion peptides and lectin. Also, studies ondistancing ligands from matrix cores have been described.

Alternative methods for purifying influenza A include, for example, sizeexclusion and ion exchange chromatographies.

SUMMARY

The present disclosure provides methods of manufacturing and usingfluid-insoluble material complexes that specifically capturemicroorganisms, proteins, and other biologicals and remove them fromfluids. It also pertains to the option of controllably releasing thecaptured biologicals under certain conditions.

Some embodiments pertain to methods of immobilizing a biological bymixing a sample containing the biological with a material complex andphysically separating the immobilized biological from the sample. Thebiological is immobilized by its adsoption to the material complex. Thephysical separation of the immobilized biological from the rest of thesample can be achieved by such methods as decantation, for examplerelying on gravity or magentic forces, and filtration. The materialcomplex can include a hydroxyl-, amino-, mercapto- or epoxy-containingmaterial that is fluid-insoluble and at least one receptor bound to thematerial. The biological can include, for example, any of a cell,tissue, tissue product, blood, blood product, protein, vaccine, antigen,antitoxin, virus, microorganism, fungus, yeast, alga, and/or bacterium.The immobilized biological can then be extracted from the materialcomplex, such as by elution. In the case of the virus, the extractedbiological can then be included in a vaccine treatment. In the case ofthe protein, the extracted biological can then be included in a vaccineor therapeutic treatment.

Some embodiments pertain to material complexes composed offluid-insoluble material and a receptor. The material can be agarose,sand, textile, or any combination thereof. The fluid-insoluble materialhas reactive functionality on its surface, including hydroxyl, amino,mercapto or epoxy functionality material. The receptor is selected fromthe group consisting of mono- and poly-saccharides, heparin, or anycombination thereof. In some embodiments, the composition can include alinker bridging between the material and the receptor. The linker iscovalently bonded to the material and the receptor. The linker can be alinear or branched small molecule or polymer.

Some embodiments pertain to methods of manufacturing a compositioncomprising attaching a receptor on a hydroxyl-containing material, anepoxy-containing material, and an amino-containing material. Thereceptor can be lactose. The material can be sand, agarose,poly(glycidyl methacrylate) (PGMA), or PGMA-NH₂.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that the featuresrecited herein, advantages and objects of the disclosure will becomeclear and can be understood in detail. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate suitable embodiments and should not be considered to limitthe scope of the disclosure.

FIG. 1 illustrates three different embodiments; fluid-insoluble materialcores are complexed with receptors either directly or indirectly throughlinkers.

FIGS. 2A and 2B illustrate material complexes for fluid disinfection.

FIG. 3 illustrates the use of material complexes in affinitychromatography to concentrate micro-organisms in solutions.

FIGS. 4A, 4B, and 4C illustrate examples of direct attachment ofreceptors to materials.

FIGS. 5A, 5B, and 5C illustrate examples of attachment of receptors tomaterials via linkers.

FIG. 6 illustrates a general route for covalent coupling when using1,1′-carbonyldiimidazole.

FIG. 7 illustrates quantification of the binding of recombinanthemagglutinin to insoluble materials in PBS buffer; quantification wasperformed using the Bradford Protein Assay by measuring absorbance at595 nm wavelength.

FIG. 8 illustrates quantification of influenza A attachment to insolublematerials.

FIG. 9 illustrates the activity of a complex of PGMA polymer whilevarying the initial titer of influenza A.

DESCRIPTION

The present disclosure is directed to synthesizing and usingfluid-insoluble material complexes that specifically capture biologicalsand remove them from fluids. It also pertains to the option ofcontrollably releasing the captured biologicals under specificconditions.

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the devices and methods disclosed herein. One ormore examples of these embodiments are illustrated in the accompanyingdrawings. Those skilled in the art will understand that the devices andmethods specifically described herein and illustrated in theaccompanying drawings are non-limiting exemplary embodiments and thatthe scope of the present disclosure is defined solely by the claims. Thefeatures illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present disclosure.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contentclearly dictates otherwise. The terms used in this disclosure adhere tostandard definitions generally accepted by those having ordinary skillin the art. In case any further explanation might be needed, some termshave been further elucidated below.

The term “biologicals” as used herein refers to living organisms andtheir products, including, but not limited to, cells, tissues, tissueproducts, blood, blood products, proteins, vaccines, antigens,antitoxins, viruses, microorganisms, fungi, yeasts, algae, bacteria,etc., or any combination thereof. One example of a biological caninclude microorganisms, such as pathogenic or non-pathogenic bacteria.Another example of biologicals can include viruses, viral products,virus-imitating entities, or any combination thereof. In someembodiments, the biological is selected from the group consisting ofcell, tissue, tissue product, blood, blood product, body fluid, productof body fluid, protein, vaccine, antigen, antitoxin, biologicalmedicine, biological treatment, virus, microorganism, fungus, yeast,alga, bacterium, prokaryote, eukaryote, Staphylococcus aureus,Streptococcus, Escherichia coli (E. coli), Pseudomonas aeruginosa,mycobacterium, adenovirus, rhinovirus, smallpox virus, influenza virus,herpes virus, human immunodeficiency virus (HIV), rabies, chikungunya,severe acute respiratory syndrome (SARS), polio, malaria, dengue fever,tuberculosis, meningitis, typhoid fever, yellow fever, ebola, shingella,listeria, Yersinia, West Nile virus, protozoa, fungi Salmonellaenterica, Candida albicans, Trichophyton mentagrophytes, poliovirus,Enterobacter aerogenes, Salmonella typhi, Klebsiella pneumonia,Aspergillus brasiliensis, and methicillin resistant Staphylococcusaureus (MRSA), or any combination thereof.

In some embodiments, fluid-insoluble materials can be complexed withmicrooganism-capturing groups (“receptors”), the structures of which aredrawn from natural cellular receptors, antibodies, or simply fromavailable data describing microoganism interaction with solublemolecules. The receptors can be directly attached to the material (FIG.1, Mode A) or through a linker (FIG. 1, Mode B). In order to protect theintegrity of the molecular structure of the subject material complexes,particularly when re-cycling is a requirement, one method ofinter-connecting the receptors, linkers and materials can be viacovalent bonding. For certain applications where added structuralstability is not needed, for example in case of single use materialcomplexes, physical bonding can substitute covalent bonding. Thereceptors play a direct role by capturing the microorganims throughphysical bonding. One role of the linkers is to position the receptorsat an active distance from the core of the material. By distancing thereceptors from the core of the material, the receptors can easily accessthe target microorganisms. Another role for the linkers, particularlywhen they are branched, is to increase the density of the receptors onthe surface of the material (FIG. 1, Mode C). Increase in the density ofreceptors correlates with an increase in the capacity of capturinghigher concentrations of microorganisms.

Representative examples of the three main components of the materialcomplexes are: 1) materials: agarose, sand, textiles (such ascellulose/cotton, wool, nylon, polyester), metallic particles (includingnanoparticles), magnetic particles (including nanoparticles), glass,fibergalss, silica, wood, fiber, plastic, rubber, ceramic, percelain,stone, marble, cement, biological polymers, natural polymers andsynthetic polymers (such as PGMA), or any combination thereof; 2)receptors: lactose (natural and synthetic) and its derivatives (such assialyllactose), mono- and poly-saccharides (natural and synthetic),heparin and chitosan, or any combination thereof; and 3) linkers: linearand branched polymers, such as poly(ethylene glycol) (PEG) andpoly(ethylenimine) (PEI, various ratios of primary:secondary:tertiaryamine groups), (e.g. multi-arm branched PEG-amines), dendrons anddendrimers (e.g. liyperbranched bis-MPA polyester-16-hydroxyl) andchitosan, or any combination thereof. Each of the material complexes mayincorporate the material and the receptor components. However,incorporating the linker component is optional.

Suitable metal materials include, but are not limited to, stainlesssteel, nickel, titanium, tantalum, aluminum, copper, gold, silver,platinum, zinc, Nitinol, Inconel, iridium, iron, tungsten, silicon,magnesium, tin, alloys, coatings containing any of the foregoing,galvanized steel, hot dipped galvanized steel, electrogalvanized steel,annealed hot dipped galvanized steel, or any combination thereof.

Suitable glass materials include, but are not limited to, soda limeglass, strontium glass, borosilicate glass, barium glass, glass-ceramicscontaining lanthanum, or any combination thereof.

Suitable sand materials include, but are not limited to, sand comprisedof silica (e.g., quartz, fused quartz, crystalline silica, fumed silica,silica gel, and silica aerogel), calcium carbonate (e.g., aragonite), orany mixture thereof. The sand can comprise other components, such asminerals (e.g., magnetite, chlorite, glauconite, gypsum, olivine,garnet), metal (e.g., iron), shells, coral, limestone, and rock, or anycombination thereof.

Suitable wood materials include, but are not limited to, hard wood andsoft wood, and materials engineered from wood, wood chips, or fiber(e.g., plywood, oriented strand board, laminated veneer lumber,composites, strand lumber, chipboard, hardboard, medium densityfiberboard). Types of wood include alder, birch, elm, maple, willow,walnut, cherry, oak, hickory, poplar, pine, fir, or any combinationthereof.

Suitable fiber materials include, but are not limited to, natural fibers(e.g., derived from an animal, vegetable, or mineral) and syntheticfibers (e.g., derived from cellulose, mineral, or polymer). Suitablenatural fibers include cotton, hemp, jute, flax, ramie, sisal, bagasse,wood fiber, silkworm silk, spider silk, sinew, catgut, wool, sea silk,wool, mohair, angora, and asbestos, or any combination thereof. Suitablesynthetic fibers include rayon, modal, and Lyocell, metal fiber (e.g.,copper, gold, silver, nickel, aluminum, iron), carbon fiber, siliconcarbide fiber, bamboo fiber, seacell, nylon, polyester, polyvinylchloride fiber (e.g., vinyon), polyolefin fiber (e.g., polyethylene,polypropylene), acrylic polyester fiber, aramid, spandex, or anycombination thereof.

Suitable natural polymer materials include, but are not limited to, apolysaccharide (e.g., cotton, cellulose), shellac, amber, wool, silk,natural rubber, and a biopolymer (e.g., a protein, an extracellularmatrix component, collagen), or any combination thereof.

Suitable synthetic polymer materials include, but are not limited to,polyvinylpyrrolidone, acrylics, acrylonitrile-butadiene-styrene,polyacrylonitrile, acetals, polyphenylene oxides, polyimides,polystyrene, polypropylene, polyethylene, polytetrafluoroethylene,polyvinylidene fluoride, polyvinyl chloride, polyethylenimine,polyesters, polyethers, polyamide, polyorthoester, polyanhydride,polysulfone, polyether sulfone, polycaprolactone, polyhydroxy-butyratevalerate, polylactones, polyurethanes, polycarbonates, polyethyleneterephthalate, as well as copolymers thereof, or any combinationthereof.

Suitable rubber materials include, but are not limited to, silicones,fluorosilicones, nitrile rubbers, silicone rubbers, polyisoprenes,sulfur-cured rubbers, butadiene-acrylonitrile rubbers,isoprene-acrylonitrile rubbers, and the like, or any combinationthereof.

Suitable ceramic materials include, but are not limited to, boronnitrides, silicon nitrides, aluminas, silicas, and the like, or anycombination thereof.

Suitable stone materials include, but are not limited to, granite,quartz, quartzite, limestone, dolostone, sandstone, marble, soapstone,serpentine, or any combination thereof.

Exemplary receptors can include: 1) heparin, a negatively chargedpolymer that can mimic innate glycosaminoglycanes found in thememebranes of host cells. It is commercially available as heparin sodiumwhich is extracted from porcine intestinal mucosa and is approved asblood anti-coagulant. Also, non-animal-derived syntheticheparin-mimicking sulfonic acid polymers can act in a similar fashion tonatural heparin; 2) chitosan, an ecologically friendly bio-pesticidethat can ligate to a variety of microorganisms and proteins. It is alsoused as a hemostatic agent and in transdermal drug delivery; and 3)lactose, a by-product of the dairy industry. It is widely available andproduced annually in millions of tons. Lactose can also be synthesizedby condensation/dehydration of the two sugars, galactose and glucose,including all their isomers.

Exemplary materials can include: 1) sand, an affordable and widelyavailable material. In addition, complexed sand could easily replacenon-complexed sand in established technologies such as drinking waterpurification; 2) agarose, particularly Sepharose®, a beadedpolysaccharide polymer extracted from seaweed. They are also widelyavailable and used in chromatography to separate biomolecules; and 3)PGMA, a synthetic polymer produced from Glycidyl methacrylate, which isan ester of methacrylic acid and a common monomer used in the productionof epoxy materials.

Exemplary linkers can include: 1) chitosan, see its description as areceptor; 2) Poly(ethylene glycol) and its derivatives, produced fromethylene oxides with many different chemical, biological, commercial andindustrial uses; and 3) dendrons and dendrimers, relatively newmolecules. They are repetitively branched molecules using a small numberof starting reagents. They are commonly used in drug delivery and insensors.

In some embodiments, the receptors can be directly attached to thematerial (FIG. 4) or through linkers (FIG. 5) via chemical coupling. Onetype of coupling reagent is 1,1′-carbonyldiimidazole (CDI). The couplingreagent may also be N,N′-Dicyclohexylcarbodiimide (DCC) orN-(3-Dimethylaminopropyl)-N′-ethylcarbodibide hydrochloride (EDC orEDCI).

An exemplary coupling reagent is CDI. Basic protonated end groups, suchas hydroxyl groups (R—OH) in sand and Sepharose® and tertiary aminegroups (R—NH₂) in PGMA-diaminobutane, readily react with CDI to form anester or amide link. The resulting imidazole-substituted derivatives arereacted with hydroxyl-terminated receptors yielding either carbonates[R—O—C(O)—O-receptor] or carbamates [R—N(H)—C(O)—O-receptor]. Theresulting imidazole-substituted derivatives can also be reacted withamine-terminated receptors yielding urea derivatives[R—N(H)—C(O)—N(H)-receptor] (FIG. 6). Due to the formation of a covalentbond between the receptor and the material (direct bonding or through alinker), the structure of the bound receptor is different compared tothe structure of the commercially available free receptor. For example,as depicted in FIG. 6, the receptor looses a hydrogen atom upon reactionwith the immidazole-substituted derivatives to form areceptor-carbonate, receptor-carbamate or receptor-urea derivative.

If an appropriate functional group is not present on the surface of thematerial, a suitable functional group can be made available of thesurface by a chemical transformation. In general, a chemicaltransformation can be hydrolysis, oxidation (e.g., using Collinsreagent, Dess-Martin periodinane, Jones reagent, and potassiumpermanganate), reduction (e.g., using sodium borohydride or lithiumaluminum hydride), alkylation, deprotonation, electrophilic addition(e.g., halogenation, hydrohalogenation, hydration), hydrogenation,esterification, elimination reaction (e.g., dehydration), nucleophilicsubstitution, radical substitution, or a rearrangement reaction. Ifneeded, more than one chemical transformation, successively orsimultaneously, can be used to provide a suitable functional group or aheterogeneous group of functional groups of various identities.Alternatively, a monomer with a desired functional group can be graftedto the material.

In some embodiments, the chemical transformation is hydrolysis.Generally, the hydrolysis is performed with water in the presence of astrong inorganic, organic, or organo-metallic acid (e.g., stronginorganic acid, such as hydrochloric acid, sulfuric acid, phosphoricacid, nitric acid, hydroiodic acid, hydrobromic acid, chloric acid, andperchloric acid) or strong inorganic, organic, or organo-metallic base(e.g., Group I and Group II hydroxides, such as lithium hydroxide,sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesiumhydroxide, magnesium hydroxide, calcium hydroxide, and barium hydroxide;ammonium hydroxide; and sodium carbonate). For example, a materialcomprising an acyl halide can undergo hydrolysis to form a carboxylicacid.

In some embodiments, the chemical transformation is a substitutionreaction where one functional group is replaced with another. Forexample, a material comprising a haloalkyl group can react with a strongbase to form a hydroxy group.

In some embodiments, the chemical transformation is alkylation,hydrogenation, or reduction. For example, a material comprising ahydroxy or haloalkyl (e.g., iodoalkyl or bromoalkyl) moiety can bereacted with ammonia to form an amino group. A material comprising ahaloalkyl moiety also can be converted to a mercapto group byS-alkylation using thiourea. A material comprising a nitrile can behydrogenated to form an amino group. A material comprising an amidogroup can be reduced (e.g., in the presence of lithium aluminum hydride)to form an amino group. A material comprising a formyl or keto group canbe reduced to form an amino or hydroxy group. Multiple homogeneous orheterogeneous transformations can be applied simultaneously orsuccessively.

The material complexes can be formed by any suitable method usingsuitable temperatures (e.g., room temperature, reflux), reaction times,solvents, catalysts, and concentrations. In some embodiments, an excessamount of linkers and receptors will be used to ensure an effectiveamount of receptors in the material complexes.

In some embodiments, attachments amongst receptors, linkers, andmaterials can be secured physically. This is achieved by mixingreceptors or linkers, or combinations thereof, dissolved in solventswith the materials, then allowing the solvents to evaporate in air orunder vacuum.

The receptors may also reversibly interact with the target biologicals,such as micro-organisms or virus. The biologicals can be desorbed fromthe receptors, such as through elution. Eluents such ashigher-then-physiological sodium chloride solutions andlactose-containing solutions are capable of desorbing the biologicalfrom the material complexes.

Depicted in FIGS. 4 and 5, one common receptor is lactose Immobilizedlactose can be used for capturing a high titer of influenza A virus.Furthermore, lactose-PGMA combination is also an exemplary material.

The material complexes can be used for the removal of biologicals fromfluids and in affinity chromatography setups. These material complexesshould not dissolve in the aforementioned fluids and are expected toreduce or eliminate the generation of harmful chemical or biologicalbyproducts. The material complexes can be used in a batch setup (FIG.2A) or in a flow setup (FIG. 2B). In a batch setup, the materialcomplexes are added to the fluids and then separated by decantation orfiltration. In a flow setup, the fluids are circulated through one ormultiple cartridges containing the material complexes. In the flowsetup, the cartridges are stopped with filters, the porosity of which issmaller than the size of the material complexes. Such small porositystops the material complexes from escaping the cartridges.

Affintity chromatography may also involve the separation and potentiallypurification of target biologicals from a complex mixture based on areversible adsorption of the desired biologicals onto thechromatographic matrix. Where the disclosed material complexes are usedto purify biologicals, such as microbes and proteins for vaccine andtherapeutic drug manufacturing, the biological-containing solutions maybe passed through the material complexes packed in a cartridge (FIG. 3).While the target biologicals are expected to bind to the materialcomplexes, the impurities are expected to pass through the cartridgewithout binding to the material complexes. The next step is the elutionof the bound target biologicals off of the material complexes usingimpurity-free eluents. Depending on the titer of the biological and itsaffinity to material complexes, the process of biological separation,purification, or combinations thereof, can be achieved by multiplecirculations through the same cartridge or passage through successivecartridges.

The disclosed methods and material complexes may be used in a number ofapplications including, for examples: 1) pharmaceuticals: purificationof vaccines, proteins, including monoclonal antibodies (MAbs), and otherbiologicals; 2) diagnostics: increasing the concentration of targetbiologicals in samples leading to increase in sensitivity in existingand novel diagnostic tools, or including materials that change colorupon binding a biological molecule allowing simple point-of-usediagnostics; 3) prophylactics: trapping biologicals prior to infectionor contamination (e.g. face masks, air purifiers, gloves); 4)therapeutics: disinfection of blood and its products, extracorporealdialysis, disinfection of intestinal fluids, and controlling thebiological composition of life-sustaining fluids; and 5) environmental:removing biologicals from water and other fluids in the environment,including air.

In some embodiments, the disclosed methods and material complexes can beused for vaccine purification. Current vaccine purification techniquesuse a combination of membrane separation, such as ultrafiltration, andchromatographic separation, such as size exclusion and ion exchange.While the overall purity is above about 90%, the yield is only about50%. The disclosed methods and material complexes can substitute theseparations based on size exclusion, ion exchange chromatography, orcombinations thereof. If the disclosed methods and material complexesshow high selectivity towards the target biologicals, then it ispossible that the disclosed methods and material complexes couldsubstitute both chromatograpic separations as well as the membraneseparation and other filtration steps, and combinations thereof.

In some embodiments, the disclosed methods and material complexes can beused for water disinfection. Current water disinfection techniques arebased on irradiation with UV light, use of ozone, or use of bleachingchemicals designed to kill viruses and bacteria. Unfortunately, the UVlight technique and generation of ozone are both very energy consuming,and the chemicals that kill viruses and bacteria are also harmful tohumans. Moreover, many of the chemical and biological by-products thatresult from these disinfection techniques can cause serious diseases,such as cancer and damage to the nervous system. The disclosed methodsand material complexes can disinfect water at a relatively low cost andwithout inducing such side effects. Two application methods can befollowed to disinfect water, batch and flow setups (FIG. 2). In the caseof water disinfection application, some exemplary receptors may beheparin and chitosan.

In some embodiments, the disclosed methods and material complexes can beused for pathogen inactivation in transfusion blood. Reduction ofinfectious entities and controlling the biological composition intransfusion blood without compromising the blood quality is the targetof various techniques, particularly the proactive pathogen inactivationtechniques. Current methods include Solvent Detergent (SD), heattreatments and use of photosensitive chemicals. However, thesetechniques induce changes in normal blood composition leading topost-transfusion side effects. For example, plasma pasteurizationdenatures clotting factors, SD treatment results in loss ofα₂-antiplasmin crucial in hemostasis, and photosensitive chemicals areimmunogenic, lower platelet count increment in recipients, and generatechemical by-products. Moreover, following deactivation of, for example,HIV-1, the basic peptides released, HIV-1′s Tat (transactivating factor)and gp120, induce AIDS-associated pathologies. The disclosed methods andmaterial complexes may result in pathogen inactivation while avoidingsuch side effects. One method may include mixing or passing a sample,such as viremic allogeneic blood or its products, or combinationsthereof, with or through the material complexes. The material complexescan be packed in a cartridge, i.e. flow setup, or be mixed with thesample, i.e. batch setup. Then, the mixture of sample and materialcomplex can be filtered through a membrane filter. Depending on theviral titer and the viral affinity: 1) the flow setup can includemultiple circulations through the same packed cartridge or passagethrough successive cartridges; and 2) the batch setup can includesuccessive mixing of the sample with multiple beds of fresh materialcomplexes. In the case of pathogen inactivation in transfusion blood,one receptor may be heparin.

In some embodiments, the disclosed methods and material complexes can beused for purification of viruses, such as an influenza vaccine, orvirus-imitating particles. The materials currently used to purifyinfluenza vaccines are either poorly selective and cheap to produce orhighly selective and expensive to produce. The former class of materialsfor vaccine purification is dominated by non-selective chemistry-basedmaterials, while the later class of materials for vaccine purificationis dominated by highly selective biology-based materials. The formernon-selective materials lead to low yield and low purity, two majorsetbacks in vaccine manufacturing. The main shortcomings of the lattergroup of materials, which include immobilized antibodies, are: 1)expense; 2) the requirement for re-qualification after each recycling;and 3) degradation of the immobilized biological molecules over time.The disclosed material complexes are inexpensive chemistry-basedmaterials that are capable of matching the selectivity to biology-basedmaterials. In the case of influenza vaccine purification applications,some receptors include lactose (natural and synthetic) and itsderivatives.

The present disclosure is also directed to kits comprising any one ormore of the material complexes disclosed herein, and/or reagents usedfor making and/or using them.

The present disclosure is also directed to products comprising any ofthe compositions disclosed herein. In some embodiments, the product isselected from a vaccine, medical device, diagnostic equipment, implant,glove, mask, textile, surgical drape, tubing, surgical instrument,safety gear, fabric, apparel item, floor, handle, wall, sink, shower,tub, toilet, furniture, wall switch, toy, athletic equipment, playgroundequipment, shopping cart, countertop, appliance, railing, door, airfilter, air processing equipment, water filter, water processingequipment, pipe, phone, cell phone, remote control, computer, mouse,keyboard, touch screen, leather, cosmetic, cosmetic making equipment,cosmetic storage equipment, personal care item, personal care itemmaking equipment, personal care storage equipment, animal care item,animal care item making equipment, animal care storage equipment,veterinary equipment, powder, cream, gel, salve, eye care item, eye careitem making equipment, eye care storage equipment, contact lens, contactlens case, glasses, jewelry, jewelry making equipment, jewelry storageequipment, utensil, dish, cup, container, object display container, fooddisplay container, food package, food processing equipment, foodhandling equipment, food transportation equipment, food storageequipment, food vending equipment, animal housing, farming equipment,animal food handling equipment, animal food storage space, animal foodprocessing equipment, animal food storage equipment, animal foodcontainer, air vehicle, land vehicle, water vehicle, water storagespace, water storage equipment, water storage container, waterprocessing equipment, water storage equipment, water filter, and airfilter, or any combination thereof.

The methods and material complexes described herein are designed tocapture complete biologicals, such as viruses, microorganisms andbacteria, and physically remove them from the original sample. Thecaptured biologicals can be released from the material complexes byelution using a release solutions or eluents capable of desorbing thecaptured biologicals from the material complexes, such ashigher-then-physiological sodium chloride solutions or lactosesolutions.

U.S. Provisional Application Ser. No. 61/784,432 filed Mar. 14, 2013 isincorporated herein by reference in its entirety.

EXAMPLES Example 1 Synthesis of Lactose-Sand (FIG. 4A)

The synthesis followed these steps: 5 grams of fine sand was rinsed with20 ml DI water while on a medium frit filter. They were then mixed with10 ml pH 8.5 (20 mM) borate buffer and allowed to stir for 10 minutes atroom temperature. Thirty nine mg of 1,1′-carbonyldiimidazole (0.24 mmol,MW 162.15) was then added to the suspension and allowed to react for 2hours before adding 190 mg of β-D-lactose (0.55 mmol). The resultingmixture was allowed to stir for 4 days at room temperature. The finalsuspension was filtered and the solid was rinsed with de-ionized (DI)water. The wetness of the solid was preserved.

Example 2 Synthesis of Lactose-Sepharose® (FIG. 4B)

The synthesis followed these steps: 5 grams of wet Sepharose (ca. 5 wt %in water) was mixed with 10 ml pH 8.5 (20 mM) borate buffer and allowedto stir for 10 minutes at room temperature. Thirty nine mg of1,1′-carbonyldiimidazole (0.24 mmol, MW 162.15) was then added to thesuspension and allowed to react for 2 hours before adding 190 mg ofβ-D-lactose (0.55 mmol). The resulting mixture was allowed to stir for 4days at room temperature. The final suspension was filtered and thesolid was rinsed with 100 ml DI water. The wetness of the solid waspreserved.

Example 3 Synthesis of Lactose-PGMA (FIG. 4C)

The synthesis followed these steps: A 100 ml single neck round bottomflask and a magnetic bar were dried under vacuum while hot. Fifty ml drytetrahydrofuran was added followed by 1.24 g (14 mmol) of1,4-diaminobutane. While stirring the solution, 200 mg PGMA (1.4 mmolequivalents of the repeat unit) was added. The solution was then allowedto stir at room temperature for 10 minutes before starting the in-situevacuation into a cold trap, using the vacuum line. The reaction flaskwas gently heated using a heating gun in order to ensure the removal ofall volatile reagents. To the resulting oil-like product, 50 ml DI waterwere added leading to the precipitation of a white film-like solid. Thissolid was then filtered on a medium frit and rinsed with 300 ml DIwater. The yield was 0.529 g of PGMA-NH₂. The final polymer wasefficiently dried and stored at low temperature.

One hundred and ten mg of the resulting intermediate, PGMA-NH₂, wasmixed with 10 ml pH 8.5 20 mM borate buffer and allowed to stir for fewminutes at room temperature. Nineteen mg of 1,1′-carbonyldiimidazole wasthen added to the suspension and allowed to stir for 1 hour beforeadding 0.055 g of β-D-lactose. The final mixture was allowed to stir fortwo days at room temperature followed by filtering through a medium fritand rinsing with 50 ml DI water. The wetness of the solid was preserved.

Example 4 Synthesis of Lactose-[branching]-Sand (FIG. 5A)

The synthesis followed these steps: Five grams of fine sand wasvigorously stirred with 20 ml DI water, then filtered through a mediumfrit. They were then mixed with 10 ml pH 8.5 20 mM borate buffer andallowed to stir for few minutes at room temperature. Sixteen mg of1,1′-carbonyldiimidazole (0.1 mmol, MW 162.15) was then added to thesuspension and allowed to stir for 2 more hours before adding 0.25 g ofHyperbranched bis-MPA polyester-16-hydroxyl (0.1425 mmol, 228 mmol. eq.OH). After two additional hours, 0.37 g (2.28 mmol) of1,1′-carbonyldiimidazole was added to the suspension and allowed to stirfor 2 more hours before adding 3.9 g (11.4 mmol) of β-D-lactose. Five mlof the pH 8.5 borate buffer was then added. The final “almost clear”mixture was allowed to stir for two days at room temperature. The finalsolution was filtered through a medium frit and rinsed with 50 ml DIwater, isolating 4.8943 g of sand complex the color of which was similarto that of the starting sand. The wetness of the solid was preserved.

Example 5 Synthesis of Lactose-[branching]-Sepharose (FIG. 5B)

The synthesis followed these steps: One gram of wet Sepharose (ca. 5 wt% in water) was mixed with 10 ml pH 8.5 20 mM borate buffer and allowedto stir for few minutes at room temperature. Thirty two mg of1,1′-carbonyldiimidazole (0.2 mmol, MW 162.15) was then added to thesuspension and allowed to stir for 2 more hours before adding 0.5 g ofHyperbranelted bis-MPA polyester-16-hydroxyl (0.285 mmol, 4.56 mmol.eq.OH). After two additional hours, 0.74 g (4.56 mmol) of1,1′-carbonyldiimidazole was added to the suspension and allowed to stirfor 2 hours before adding 7.8 g (22.8 mmol) of β-D-lactose. Additional 5ml of the pH 8.5 buffer was added. The final white mixture was allowedto stir for two days at room temperature. Fifty ml DI water were addedto the final dense white solution to ensure dissolution of all freereagents. The final solution was filtered through a medium frit andrinsed with 50 ml DI water. The wetness of the solid was preserved.

Example 6 Synthesis of Lactose-[branching]-PGMA (FIG. 5C, 1)

The synthesis, including a dendrimer, followed these steps: One hundredmg of PGMA-NH₂ (0.4 mmol equivalents of the repeat unit) was mixed with50 ml pH 8.5 20 mM borate buffer and allowed to stir for few minutes atroom temperature. Sixty four mg of 1,1′-carbonyldiimidazole (0.4 mmol,MW 162.15) was then added to the suspension and allowed to stir for 2hours before adding 1 g of Hyperbranched bis-MPA polyester-16-hydroxyl(0.57 mmol, 9.12 mmol.eq. OH). After two additional hours, 1.48 g (9.12mmol) of 1,1′-carbonyldiimidazole was added to the suspension andallowed to stir for 2 more hours before adding 15.6 g (45.6 mmol) ofβ-D-lactose. The final white mixture was allowed to stir for two days atroom temperature. Fifty ml DI water was added to the final dense whitesolution to ensure dissolution of all free reagents. The final solutionwas filtered through a medium frit and rinsed with 50 ml DI water. Thewetness of the solid was preserved.

Example 7 Synthesis of Lactose-[branching]-PGMA (FIG. 5C, 2)

The synthesis, including chitosan, followed these steps: Four hundred mlof 0.5% acetic acid in DI water was prepared by adding 2 g of the acidto 400 mL of water. To this acid solution, 2 g of Chitosan was added andthe solution was allowed to stir at room temperature for 5 minutes untilbecoming monophasic. Then, 200 mg of PGMA was added and the finalsuspension was allowed to stir at room temperature for two hours. Thefinal off-white suspension was then filtered through a medium frit andthe solid was washed with 100 ml of DI water. The isolated solid wasre-suspended in 10 ml DI water. Its pH was ca. 4. One drop of a sodiumcarbonate solution (5 wt % sodium carbonate solution prepared bydissolving 500 mg of Na₂CO₃ in 9.5 g DI water) was added to increase thepH to ca. 9. The now basic mixture was filtered and rinsed with 50 ml DIwater. The yield was 140 mg of chitosan-PGMA. One hundred mg of thisintermediate was suspended in 10 ml pH 8.0 borate buffer. 0.148 g (0 9mmol) of 1,1′-carbonyldiimidazole was added to the suspension andallowed to stir for 2 hours before adding 1.56 g (4.5 mmol) ofβ-D-lactose. The final mixture was allowed to stir for two days at roomtemperature. The final solution was filtered through a medium frit,rinsed with 100 ml DI water.

Example 8 Synthesis of Lactose-[branching]-Sand

The synthesis will follow these steps: Five grams of fine sand will bevigorously stirred with 20 ml DI water, then filtered through a mediumfrit. They will then be mixed with 10 ml pH 8.5 20 mM borate buffer andallowed to stir for few minutes at room temperature. Sixteen mg of1,1′-carbonyldiimidazole (0.1 mmol, MW 162.15) will then be added to thesuspension and allowed to stir for 2 more hours before adding branchedpoly(ethylene glycol) (2.28 mmol.eq. OH). After two additional hours,0.37 g (2.28 mmol) of 1,1′-carbonyldiimidazole will be added to thesuspension and allowed to stir for 2 more hours before adding 3.9 g (114 mmol) of β-D-lactose. Five ml of the pH 8.5 borate buffer will then beadded. The final mixture will be allowed to stir for two days at roomtemperature. The final solution will be filtered through a medium fritand rinsed with 50 ml DI water. The wetness of the solid will bepreserved.

Example 9 Synthesis of Lactose-[branching]-Sepharose

The synthesis will follow these steps: One gram of wet Sepharose (ca. 5wt % in water) will be mixed with 10 ml pH 8.5 20 mM borate buffer andallowed to stir for few minutes at room temperature. Thirty two mg of1,1′-carbonyldiimidazole (0.2 mmol, MW 162.15) will then be added to thesuspension and allowed to stir for 2 more hours before adding branchedpoly(ethylene glycol) (4.56 mmol.eq. OH). After two additional hours,0.74 g (4.56 mmol) of 1,1′-carbonyldiimidazole will be added to thesuspension and allowed to stir for 2 hours before adding 7.8 g (22.8mmol) of β-D-lactose. Additional 5 ml of the pH 8.5 buffer will beadded. The final mixture will be allowed to stir for two days at roomtemperature. Fifty ml DI water will be added to the final solution toensure dissolution of all free reagents. The final solution will befiltered through a medium frit and rinsed with 50 ml DI water. Thewetness of the solid will be preserved.

Example 10 Synthesis of Lactose-[branching]-PGMA

The synthesis, including a branched polymer, will follow these steps:One hundred mg of PGMA-NH₂ (0.4 mmol equivalents of the repeat unit)will be mixed with 50 ml pH 8.5 20 mM borate buffer and allowed to stirfor few minutes at room temperature. Sixty four mg of1,1′-carbonyldiimidazole (0.4 mmol, MW 162.15) will then be added to thesuspension and allowed to stir for 2 hours before adding branchedpoly(ethylene glycol) (9.12 mmol.eq.OH). After two additional hours,1.48 g (9.12 mmol) of 1,1′-carbonyldiimidazole will be added to thesuspension and allowed to stir for 2 more hours before adding 15.6 g (456 mmol) of β-D-lactose. The final mixture will be allowed to stir fortwo days at room temperature. Fifty ml DI water will be added to thefinal solution to ensure dissolution of all free reagents. The finalsolution will be filtered through a medium frit and rinsed with 50 ml DIwater. The wetness of the solid will be preserved.

Example 11 Influenza-A Virus

Since influenza's envelope protein, hemagglutinin (HA), is known tostrongly bind to innate sialic acid in membranes of host cells,covalently attaching sialyllactose onto insoluble supports would allowvirus adsorption to these surfaces. Thus, sialyllactose-complexed withPGMA was prepared following FIG. 5C, 2 using 6′-sialyllactose instead ofβ-D-lactose as the starting material. The linker therein was chitosan.Chemical derivatization of the material was monitored by recombinant HAbinding assays (quantified by the Bradford test) (FIG. 7).

The PGMA-attached sialyllactose along with a set of controls were testedin a buffered (PBS) aqueous solution of PR8 strain of influenza-A virus,with the viral titers in the supernatants quantified using the plaqueassay. The results revealed that PGMA-chitosan-lactose removed more than98% of the virus from solution (Table 1 and FIG. 8). Furthermore, datashowed that the virus adsorption to the disclosed material complexesfollows a linear isotherm; the relatively constant percentage ofadsorbed influenza A to the material complexes reflects Freundlichisotherm that describes adsorption of entities on suspended surfaces atvery low surface coverage. Indeed, the linearity between log (adsorbedvirus) and log (initial virus) was confirmed by obtaining a R²coefficient=0.994 (Table 2 and FIG. 9).

TABLE 1 Quantification of influenza A attachment to insoluble materialsAverage # Captured of virus in [virus] % supernatant Standard comparedMaterial (×10{circumflex over ( )}3 pfu/ml) Deviation to PBS PGMA 7.71.1 45 PGMA-Ch 8.7 2.3 38 PGMA-Ch-L 0.4 0.2 97 PGMA-Ch-SL 1.2 0.2 91 Ch12.7 2.1 9 PBS, no material 14 0.8 0 PGMA = poly(glycidyl methacrylate),Ch = chitosan, SL = sialyllactose, L = lactose

TABLE 2 Activity of complexed poly(glycidyl methacrylate) polymer whilevarying the initial titer of influenza A Starting [Virus] Adsorbed[Virus] (pfu/ml) (pfu/mg) % Adsorbed [virus] 1,433,333 142133 99.228,333 2791 98.5 863 85 98.8 18000 1100 93.9

One skilled in the art will appreciate further features and advantagesof the disclosure based on the above-described embodiments. Accordingly,the disclosure is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

The invention claimed is:
 1. A method for immobilizing a biologicalcomprising: mixing a fluid sample comprising the biological through amaterial complex comprising at least one of a hydroxyl-, amino-,mercapto or epoxy-containing material that is fluid-insoluble and atleast one receptor selected from lactose, lactose derivative, mono- orpoly-saccharide, heparin, chitosan, or any combination thereof, whereinthe receptor is bound to the material; and separating the biologicalfrom the sample by adsorbing the biological to the material complex,wherein the receptor is bound to the material via a linker, wherein thelinker is a repetitively branched molecule, and wherein saidrepetitively branched molecule is any of a branched poly(ethyleneglycol) (PEG) and a branched poly(ethylenimine) (PEI) or a combinationthereof.
 2. The method of claim 1, wherein the biological is selectedfrom the group consisting of cell, tissue, tissue product, blood, bloodproduct, body fluid, product of body fluid, protein, vaccine, antigen,antitoxin, biological medicine, biological treatment, virus,microorganism, fungus, yeast, alga, bacterium, prokaryote, eukaryote,Staphylococcus aureus, Streptococcus, Escherichia coli (E. coli),Pseudomonas aeruginosa, mycobacterium, adenovirus, rhinovirus, smallpoxvirus, influenza virus, herpes virus, human immunodeficiency virus(HIV), rabies, chikungunya, severe acute respiratory syndrome (SARS),polio, malaria, dengue fever, tuberculosis, meningitis, typhoid fever,yellow fever, ebola, shigella, listeria, yersinia, West Nile virus,protozoa, fungi, Salmonella enterica, Candida albicans, Trichophytonmentagrophytes, poliovirus, Enterobacter aerogenes, Salmonella typhi,Klebsiella pneumonia, Aspergillus brasiliensis, and methicillinresistant Staphylococcus aureus (MRSA), or any combination thereof. 3.The method of claim 1, wherein the material is selected from the groupconsisting of agarose, sand, textiles, metallic particles (includingnanoparticles), magnetic particles (including nanoparticles), glass,fiberglass, silica, wood, fiber, plastic, rubber, ceramic, porcelain,stone, marble, cement, biological polymers, natural polymers, andsynthetic polymers, or any combination thereof.
 4. The method of claim1, wherein the inter-bonding between any combination of receptor,material and the linker is achieved using chemical coupling reagents orphysical non-chemical attachment, or a combination of chemical andphysical attachments.
 5. The method of claim 4, wherein the couplingreagents are selected from 1,1′-carbonyldiimidazole (CDI),N,N′-Dicyclohexylcarbodiimide (DCC), andN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC orEDCI), or any combination thereof, and wherein the physical non-chemicalattachment is achieved by deposition of receptor molecule, linkermolecule, or combination thereof, onto the material.
 6. The method ofclaim 1, wherein the material is chemically functional and the chemicalfunctionality is amino, ammonium, hydroxyl, mercapto, sulfone, sulfinicacid, sulfonic acid, thiocyanate, thione, thial, carboxyl, halocarboxy,halo, imido, anhydrido, alkenyl, alkynyl, phenyl, benzyl, carbonyl,formyl, haloformyl, carbonato, ester, alkoxy, phenoxy, hydroperoxy,peroxy, ether, glycidyl, epoxy, hemiacetal, hemiketal, acetal, ketal,orthoester, orthocarbonate ester, amido, imino, imido, azido, azo,cyano, nitrato, nitrilo, nitrito, nitro, nitroso, pyridinyl, phosphinyl,phosphonic acid, phosphate, phosphoester, phosphodiester, boronic acid,boronic ester, borinic acid, borinic ester, or any combination thereof.7. The method of claim 6, wherein the epoxy-containing material isPoly(glycidyl methacrylate) (PGMA) and the amino-containing material isPGMA-NH2.
 8. The method of claim 6, wherein the hydroxyl, mercapto, oramino group is formed on the surface by modifying the substrate by achemical transformation comprising a hydrolysis reaction with acid,base, or a combination thereof.
 9. The method of claim 1, furthercomprising physically separating the immobilized biological from thesample by filtration or decantation, by applying gravity or magneticforces, or any combination thereof.
 10. The method of claim 1, furthercomprising releasing the immobilized biological from the materialcomplex by temperature or irradiation or mechanical or thermodynamic orthermomechanic variations, or any combination thereof; or wherein theimmobilized biological is released from the material complex byvariations in pH values, concentration of chemicals, ions, sodiumchloride, or any combination thereof.
 11. A method for immobilizing abiological comprising: mixing a fluid sample comprising the biologicalthrough a material complex comprising at least one of a hydroxyl-,amino-, mercapto or epoxy-containing material that is fluid-insolubleand at least one receptor selected from lactose, lactose derivative,mono- or poly-saccharide, heparin, chitosan, or any combination thereof,wherein the receptor is bound to the material; and separating thebiological from the sample by adsorbing the biological to the materialcomplex, wherein the receptor is bound to the material via a linker,wherein the linker is a hyperbranched bis-MPA polyester-16-hydroxyl.